ABSTRACT The goal of the proposed research is to identify molecular metabolic signals of microbial syntrophy in the human gut, with the long-term goal of developing a predictive model relating host diet to healthy gut microbiota resiliency. Specifically, the research in the project leader's laboratory aims to identify and characterize the physiological function of metabolites and genes produced by human symbiotes Bacteroides thetaiotaomicron (B. theta), a bacterium, and Methanobrevibacter smithii (M. smithii), a methane-producing archaeon, in a novel co-culture system. B. theta and M. smithii are the dominant bacterium and archaeon in the human gut, respectively; their metabolisms are interdependent; and both are known to associate with gut epithelia. Perturbation of Bacteroides and/or Methanobrevibacter populations is associated with obesity, anorexia, irritable bowel disease, Crohn's disease, colorectal cancer, and diverticulosis. For these reasons, the project leader hypothesizes that metabolic syntrophy between both organisms is essential for healthy gut function and diet or pathogen challenge disrupts syntrophy, leading to digestive disorders and infectious disease. Three specific aims are proposed to test this hypothesis using a novel co-culture system. In Specific Aim 1, the project leader will optimize a B. theta/M. smithii co-culture system. Specifically, she will use anaerobic microbiology techniques to define the nutritional requirements of a syntrophic continuous co-culture in a chemostat and test nutritional enhancements under conditions encountered in the human gut. She will also use optical and confocal fluorescence microscopy to study the spatial organization (planktonic or associated in aggregates) of both organisms in co-culture. In Specific Aim 2, the project leader will create an integrated metabolic model of co-culture syntrophy based on metabolomic and transcriptomic data. Specifically, she will use next-generation sequencing technology (RNAseq) to identify gene transcripts upregulated by both organisms in syntrophic co-culture to improve accuracy of the metabolic system models. The system model will be validated with global and targeted metabolomics data. The refined system model, integrating transcriptomic and metabolomics datasets, will be used to predict how pathogen challenge affects syntrophic B.theta/M.smithii metabolism. Finally, in Specific Aim 3, the project leader will test co-culture resiliency to pathogen challenge. She will measure the metabolic and transcriptional changes that occur in the co-culture system when challenged by the mouse gut pathogen enterohemorrhagic Citrobacter rodentium. These experiments will be used to further refine and test the system model generated in Specific Aim 2. The innovative outcome of this research will be the demonstration that B. theta and M. smithiii in co-culture communicate through the two-way exchange of small molecule metabolites. Addition of a pathogen is expected to disrupt this communication system. This work is highly significant in that it will demonstrate syntrophy between common gut microbes that contribute to human health and disease.